The long-term goal of this project is to understand how cellular and tissue-tissue interactions regulate development of the central nervous system. Disruption of tissue-tissue interactions can lead to a wide range of serious birth defects affecting the brain and eye, such as holoprosencephaly, congenital anophthalmia, microphthalmia, and anencephaly. Understanding the normal process of development will allow for better prevention and treatment of such defects. It is proposed to study the role of signaling molecules involved in these interactions using the embryonic eye as a model system for forebrain development, where the same mechanisms most likely regulate regional specification. Classical embryological studies have shown that the extraocular tissues are required for normal eye growth and differentiation. At present there is little information about the signals involved in these interactions. In explant cultures of optic vesicles from chick embryos, removal of the extraocular mesenchyme severely interferes with the formation of the retinal pigmented epithelium (RPE). The TGFbeta family member activin has been shown to be a candidate signal that exactly mimics the effects of the extraocular mesenchyme on RPE development in vitro. It is proposed to test the hypothesis whether the activin signaling pathway is required for RPE formation in the chick embryonic eye (Aim 1). To interfere with the activin signaling pathway in the developing RPE, soluble activin type II receptors will be applied as well as ectopic expression of antagonistic Smads (Smad6, 7) and truncated activin type II receptors using electroporation. It will be determined whether activin or a related signal is produced by the extraocular mesenchyme (Aim 2). Since cranial mesenchyme contains the inducer of RPE development, sufficient amounts of this tissue can be isolated for a degenerate PCR strategy. Subsequently, the identified molecule will be cloned and tested for the RPE-promoting activity in explant cultures and by transfection of chick embryos.